Construction of a XylS/Pm expression vector for use in gram positive hosts

Abstract

There were constructed five shuttle vectors containing the xylS/Pm cassette controlling a beta-lacatamse(bla) or luciferase (luc) gene to test the functionality of the XylS/Pm system in following gram positive hosts: Enterococcus faecium, Enterococcus faecalis, Lactococcus Lactis and Corynebacteriumglutamicum. The constructs were successfully established in the hosts by electroporation and XylS/Pmfunctionality was determined by ampicillin (amp) susceptibility, luciferase (luc) activity and real-timePCR. None of the hosts harboring constructs with xylS/Pm cassette controlling bla showed any ampresistance when induced. Only C. glutamicum harboring a construct with xylS/Pm cassette controlling lucshowed luc activity when induced. Only C. glutamicum harboring pATX5 was tested for luc transcriptionby real time PCR. The real time PCR showed that luc and xylS was expressed, but no level of difference between induced or uninduced samples was observed. Based on these results, the Pm promotor is determined to be functioning in C. glutamicum, but system proved not to be inducible. In addition to mentioned hosts, constructs were also tested in Mycobacterium smegmatis by Marte Singsås Dragset.